Glutathione is a cellular tripeptide (.gamma.-glutamylcysteinylglycine) which is perhaps the most abundant amino acid derivative contained in the cells of higher life forms. The middle amino acid in glutathione, cysteine, has a free thiol group which can compete with the nucleophilic site on nucleotide bases for reaction with electrophiles. Within the cell, glutathione functions so as to conjugate to xenobiotic toxic molecules in general, and electrophiles in particular, to render the toxic molecules less reactive against cellular macromolecules and to target the toxic molecules for subsequent metabolic and excretion pathways. The reactivity of glutathione to electrophilic molecules is facilitated by the enzyme glutathione S-transferase.
The glutathione S-transferase family of enzymes are thus responsible for the detoxification of a broad class of electrophiles and alkylating chemical agents. The glutathione S-transferase (GST) enzymes catalyze the conjugation of glutathione to a variety of compounds to create the products which are less reactive, more hydrophilic, and thus more easily excreted from the cells. The cytosolic glutathione S-transferase are known to belong to four classes, designated Alpha, Mu, Pi and Theta. A fifth class of glutathione S-transferases is a microsomal enzyme found primarily in liver endoplasmic reticulum. Higher cells each contain a family of many isozymes in each class with broad, yet overlapping, specificity. The enzyme family is believed to be one of the most important in the detoxification of reactive electrophiles within living cells.
Much recent effort has been focused on the elucidation of the tertiary structure of glutathione S-transferase so as to identify the active sites of the enzymatic molecule. It is known that the molecule binds quite specifically and with high affinity to glutathione, but binds promiscuously to a wide variety of xenobiotic, electrophilic, and alkylating chemical agents. Each of the enzymes of the four main cytosolic classes is found in dimeric form with two active sites per dimer each of which functions independently of the other. The active site has been characterized as consisting of a glutathione binding region (designated the G-site) and a non-specific hydrophobic binding region (designated the H-site) to accommodate the electrophilic substances.
The mechanism by which the enzyme enhances the nucleophilic reactivity of glutathione is poorly understood. Mechanisms have been proposed as to how that binding might occur, but the exact mechanism of enzymatic activity on the two substrates is, at this time, obscure. Similarly, while the binding specificity and the binding region to glutathione have been relatively well characterized, the nature, characteristics and binding specificity to the electrophile are less well understood.
One of the class of electrophilic compounds that are substrates for the glutathione S-transferase enzymes is the group of alkylating agents used in antineoplastic therapy. A common problem that is observed in modern cancer chemotherapy is the appearance of chemotherapeutic resistant tumor cells that, because of the resistivity, no longer respond appropriately to the antineoplastic agents. This resistance is often observed with many drugs that have no physical or mechanistic similarities to the original agent. The phenomenon, referred to as multi-drug resistance, has complicated attempts at cancer therapy. One common origin for the problem appears to be an increase in the expression of p-glycoprotein, a membrane protein pump which excretes large hydrophobic and toxic compounds from the cell. It has been demonstrated, in at least one instance, that a resistant population of malignant cells was shown to have a modified pattern of total glutathione S-transferase activity. A resistant population of MCF-7 breast cancer cells, identified through selection in adriamycin by Batist et al., J. Biol. Chem., 261:15544-15549 (1986) resulted in a subset of cells which were approximately 200 fold more resistant than the parental cells. The resistant cells were found to exhibit a 45 fold increase in total glutathione S-transferase activity, the increase being due to the result of an appearance of an isozyme not expressed in the parental cell line. Previous experiments have demonstrated that an increase in glutathione S-transferase alone, an increase conditioned by the transformation of susceptible cells with a foreign DNA construct expressing the wild-type glutathione S-transferase coding region, could increase the resistance of cells to an antineoplastic agent. As reported in Puchalski and Fahl, Proc. Natl. Acad. Sci. USA, 87:2443-2447 (1990), expression of the rat 1-1, 3-3 and the human P1-1 isozymes of glutathione S-transferase in COS cells increased their resistance to the agent. The cells were then incubated with monochlorobimane, a compound that fluorescens upon conjugation with glutathione. Fluorescence cell sorting was used to isolate populations of cells that expressed the recombinant glutathione S-transferase, and that expression was shown to confer significant resistance to alkylating agents.
A problem in the use of glutathione S-transferase as a potential genetic transformation agent to imbue cells with resistance to antineoplastic or alkylating agents is the relatively non-specific targeting of the glutathione S-transferase enzyme to the electrophilic substrate. In view of the lack of data and lack of characterization as to the binding affinity and parameters of glutathione S-transferase to the electrophilic substrates, it was not known if modifications to the molecule could be made which might enhance or alter the binding specificity or reactivity of glutathione S-transferase to xenobiotic or antineoplastic agents.